Modulation of the stability of rabbit skeletal muscle myosin light chain kinase through the calmodulin-binding domain.
The binding of Ca2+(4).calmodulin (CaM) to rabbit skeletal muscle myosin light chain kinase (MLCK) is required for expression of the enzyme's activity. While both MLCK and CaM were stable at 30 degrees C, their complex was not. The binding of CaM to MLCK resulted in a time- and temperature-dependent inactivation that reflected an intrinsic instability of the complex. Separation of the components of the inactive complex yielded functional CaM, but catalytically inert MLCK, indicating that the site of the inactivating event was confined to MLCK. The behavior of proteolytic fragments further localized this event to the C-terminal 60% of the 603-residue protein. Changes in the tryptophan fluorescence and proteolytic susceptibility of MLCK-CaM indicated that a conformational change accompanied, and thus may have caused, inactivation. Substrates protected against inactivation, as did millimolar concentrations of Mg2+, Mn2+, and Ca2+. These metals appeared to bind to a site on MLCK distinct from that which recognized Mg2+.ATP. A proteolytic fragment of MLCK lacking the ability to bind CaM, C beta 35 (residues 255-584; Edelman, A. M., Takio, K., Blumenthal, D. K., Hansen, R. S., Walsh, K. A., Titani, K., and Krebs, E. G. (1985) J. Biol. Chem. 260, 11275-11285), was unstable at 30 degrees C, whereas a similar fragment which does bind CaM, T beta 40 (residues 236-595; Edelman, A. M., Takio, K., Blumenthal, D. K., Hansen, R. S., Walsh, K. A., Titani, K., and Krebs, E. (1985) J. Biol. Chem. 260, 11275-11285), was unstable only when CaM was bound.
Kennelly
P J
PJ
Howard Hughes Medical Institute, Seattle, Washington.
Starovasnik
M A
MA
Edelman
A M
AM
Krebs
E G
EG
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002413:Cations, Divalent; D004789:Enzyme Activation; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D010449:Peptide Mapping; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology; D014364:Tryptophan
D002147:Calmodulin; D002413:Cations, Divalent; D014364:Tryptophan; D009219:Myosin-Light-Chain Kinase; D014357:Trypsin; D009218:Myosins; D002118:Calcium